None.
(1) Field of the Invention
The present invention relates to an alkaloid compound that inhibits biosynthesis of particular products of secondary metabolism. In particular, the present invention relates to an alkenylene piperidine amide wherein the alkenylene alkenylene with one or more double bonds, which can be isolated from Piper nigrum, that inhibits transcription of fungus genes nor-1, tri5, ver-1, verA, fas-1a, omt-1, alfR and ipnA. The present invention further relates to a method for identifying compounds that inhibit the biosynthesis of mycotoxins in fungi. In particular, a method for identifying compounds that inhibit biosynthesis of aflatoxin in Aspergillus spp. and deoxynivalenol in Gibberella spp.
(2) Description of Related Art
Mycotoxins are a group of structurally heterogeneous secondary metabolites produced by a diverse group of fungal plants pathogens. Infestation of crops and food commodities by mycotoxin producing fungi is a serious problem in view of the immunosuppressive, carcinogenic, cytotoxic, and teratogenic effects of the compounds in humans and animals. One of the most economically important mycotoxins worldwide is aflatoxin, a polyketide produced by several Aspergillus spp. Aflatoxin is the best studied of the mycotoxins and much of the molecular biology of the biosynthetic pathway has been determined in Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nidulans. Aspergillus flavus produces aflatoxin B1 and aflatoxin B2 whereas Aspergillus parasiticus produces in addition aflatoxin G1 and G2. Aspergillus nidulans, which is not considered to be an agricultural threat, has been used as a model genetic system for studies of aflatoxin biosynthesis because it produces sterigmatocystin, an aflatoxin precursor. The genes for aflatoxin biosynthesis are clustered in all three species. The molecular biology of aflatoxin biosynthesis is reviewed by Trail et al., in Microbiol. 141: 755-765 (1995). Aspergillus flavus and Aspergillus parasiticus are weak pathogens of corn, cotton, peanut, and nut crops: their effect is limited to a slight reduction in crop yield. However, the significant consequence of crops infected with either of these fungi is contamination by aflatoxin, which is produced under certain conditions during the infection. Traditional control strategies such as breeding crops for resistance to the fungi or chemical treatments of crops to prevent infection by the fungi have not been effective.
Aflatoxin is a secondary metabolite that appears to be the most potent naturally occurring carcinogen known (Council for Agricultural Science and technology (CAST), 1989). It is suspected of being responsible for the high incidence of human liver cancer in many areas of the world (Eaton and Gallagher, Ann. Rev. Pharmacol. Toxicol. 34: 135-139 (1994)). Aflatoxin is introduced into the food chain by preharvest and postharvest contamination of foods and feeds. Also, products from animals that have been fed aflatoxin contaminated feed may also become contaminated. Currently, the U.S. Food and Drug Administration limits the allowable amount of aflatoxin in food to 20 ppb, with slightly higher levels allowed in feeds. Because the level of aflatoxin in products destined for human consumption is strictly regulated in the U.S., aflatoxin contamination is primarily of economic importance. However, even though aflatoxin levels in foods is limited to 20 ppb, the effect of chronic exposure to low levels of aflatoxin on human health is unknown. Thus, some European countries require the presence of aflatoxin in foods intended for human consumption to be 0 ppb. In areas of the world where regulations do not exist, aflatoxin is a serious health problem (CAST, 1989).
Approaches to control of aflatoxin have been broadly grouped into preharvest and postharvest strategies. Proper grain storage can greatly reduce contamination postharvest, and some decontamination methods, while costly, are used, e.g., ammoniation. However, most research efforts at control of aflatoxin has been directed at the preharvest elimination of infection and contamination, since the ability to control preharvest contamination would reduce the need for postharvest elimination. Preharvest methods have included agricultural practices such as irrigation strategies designed to eliminate stress to crops associated with drought, which appears to increase production of aflatoxin by the fungus. Other methods include using regionally adapted varieties of crop plants. However, these methods have been expensive to implement and have not been completely effective. Chemical control methods have also been ineffective at controlling infection by these fungi.
The development of host plants that are resistant to Aspergillus infection and aflatoxin contamination has not been as successful as have programs for breeding resistance to other pathogens. In general, the resistant varieties that have been made are unstable from growing season to growing season and from region to region. Also, screening plants for resistance to colonization by Aspergillus spp. and aflatoxin contamination has been difficult. In corn, and frequently in cotton, inoculation methods have been difficult, often requiring wounding the plant to introduce the fungus, which may overwhelm the plants natural resistance reactions making it difficult to evaluate the plants resistance mechanisms (Cotty, Plant Dis. 73: 489-492 (1989)).
Methods have been developed for inhibiting mycotoxin production in crops. For example, U.S. Pat. No. 5,942,661 to Keller discloses a method of inhibiting mycotoxin production by introducing into the plant a gene encoding a lipoxygenase pathway enzyme of the mycotoxin. The method may produce transgenic plants that are substantially resistant to mycotoxin contamination. Mycotoxin resistance is further increased by introducing into the plant antisense genes for the 9-hyperoxide fatty acid producing lipoxygenases. However, reducing aflatoxin contamination by making transgenic plants resistant to aflatoxin production is expensive and time consuming, and since transformation efficiencies varies from plant species to plant species, the method may not be successful for all plant species. Furthermore, the long-term effect of introducing transgenic plants into the environment is unknown.
Since traditional methods for controlling fungal infection and/or production of aflatoxin by breeding, chemicals, or transgenic plants have not been completely effective, there is a need for an inexpensive and effective method for either controlling infection of crops by fungi such as Aspergillus spp. or Gibberella spp., or controlling the biosynthesis and accumulation of mycotoxins such as aflatoxin or deoxynivalenol in plants infected with fungi such as Aspergillus spp or Gibberella spp., respectively. There is also a need for a rapid and inexpensive method for identification of chemicals or compounds in natural extracts that inhibit production of mycotoxins such as aflatoxin and deoxynivalenol.
The present invention provides a substantially pure alkaloid compound that inhibits the biosynthesis of particular products of secondary metabolism. In particular, the present invention provides an alkenylene piperidine amide, which can be isolated from Piper nigrum. The alkenylene piperidine amide inhibits transcription from the nor-1 promoter, the tri5 promoter, ver-1 promoter, the verA promoter, the omt-1 promoter, the fas-1a promoter, alfR promoter, the ipna promoter, and mutant thereof. In a preferred embodiment the alkenylene piperidine amide inhibits at least transcription of the nor-1 promoter of Aspergillus parasiticus, the tri5 promoter of Gibberella pulicaris or the ver-1 promoter of Aspergillus nidulans without killing the fungus in vitro. In a preferred embodiment, the alkenylene is a C18 alkenylene with one or more double bonds. Preferably, the C18 alkenylene has two to four double bonds.
The compound of the present invention is useful for inhibiting biosynthesis of a mycotoxin by a fungus growing on a plant material. In particular, the compound inhibits the biosynthesis of aflatoxin and deoxynivalenol. Thus, the present invention provides a formulation which comprises as an active ingredient an alkenylene piperidine amide wherein the alkenylene is a C18 alkenylene with one or more double bonds, or its salt, or its ester, associated with one or more acceptable carriers, excipients or vehicles therefore. Preferably, the C18 alkenylene has two to four double bonds.
Thus, the present invention provides the use of a compound, which is an alkenylene alkenylene piperidine amide, wherein the alkenylene is a C18 alkenylene with one or more double bonds for treatment of plant material to inhibit mycotoxin production by a fungus. Further, the present invention provides for use of the above compound for the preparation of a composition for treatment of a plant material to inhibit mycotoxin production by a fungus. In particular, wherein the plant material is selected from the group consisting of seeds, nuts, and animal feeds.
The present invention further provides a method for inhibiting mycotoxin biosynthesis by a fungus in a plant material comprising applying an effective amount of an alkenylene piperidine amide in a carrier to the plant material wherein the compound inhibits biosynthesis of the mycotoxin. In a preferred embodiment, the alkenylene is a C18 alkenylene with one or more double bonds, preferably, two to four double bonds. In the method, the alkenylene piperidine amide can be provided in the carrier at a concentration between about 1 and 100 xcexcg/ml. In a preferred application, the plant material is selected from the group consisting of seeds, nuts, grains, and animal feeds.
Because the alkaloid of the present invention is able to inhibit the biosynthesis of mycotoxins, it would be useful to provide transgenic plants that are able to synthesize the alkaloid of the present invention. Therefore, the present invention further provides a transgenic plant that contains DNA comprising genes that encode enzymes involved in biosynthesis of the alkenylene piperidine amide wherein the compound synthesized by the transgenic plant inhibits biosynthesis of the mycotoxin by the fungus. In a preferred embodiment, transgenic plant produces an alkenylene piperidine amide wherein the alkenylene is a C18 alkenylene with one or more double bonds, preferably, two to four double bonds.
The present invention further provides a method for identifying compounds that inhibit biosynthesis of a product of secondary metabolism such as a mycotoxin in a fungus. In particular, a method is provided for identifying compounds that inhibit biosynthesis of aflatoxin by Aspergillus spp. and biosynthesis of deoxynivalenol by Gibberella spp.
Therefore, the present invention provides a method for determining whether a compound inhibits biosynthesis of a secondary metabolite comprising providing a culture of a transgenic fungus comprising a reporter gene operably linked to a promoter for a gene involved in the biosynthesis of the secondary metabolite, providing to the culture the compound to be determined, incubating the culture containing the extract under conditions that cause biosynthesis of the secondary metabolite, and measuring expression of the reporter gene wherein absence of expression of the reporter gene indicates that the compound inhibits biosynthesis of the secondary metabolite.
The present invention further provides a method for identifying and isolating a compound in a material that inhibits the biosynthesis of a secondary metabolite of a fungus comprising providing an extract of the material, separating the material into fractions or compounds by a chromatography method, providing a spore suspension of a transgenic fungus comprising a reporter gene operatively linked to a promoter that is the same as the promoter that controls transcription of a gene involved in biosynthesis of the secondary metabolite, adding the spore suspension to the separated compounds, allowing the spores to germinate and grow fungi, and detecting expression of the reporter gene wherein absence of expression of the reporter gene identifies the fractions or compounds that inhibits biosynthesis of the secondary metabolite. In a preferred embodiment of the method, the chromatography is thin layer chromatography (TLC) using TLC plates. Preferably, to grow the fungi the TLC plates are incubated in a dark moist atmosphere at 30xc2x0 C. for a time sufficient for the fungi to cover the plate. It is further preferable that the fungi be lysed by freeze-thawing to release the reporter expression product.
In a preferred embodiment of the present invention, the secondary metabolite is a mycotoxin, preferably selected from the group consisting of aflatoxin, deoxynivalenol, or sterigmatocystin. In practicing the present invention, it is preferable that the transgenic fungus be a fungus selected from the group consisting of Aspergillus parasiticus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus flavus, Gibberella pulicaris, and Gibberella zeae. In the present invention, it is preferable that the reporter gene be operably linked to a promoter that is selected from the group consisting of nor-1 promoter, ver-1 promoter, verA promoter, fas-1a promoter, omt-1 promoter, alfr promoter, ipna promoter, tri5 promoter, and mutant thereof. In an embodiment further still, it is preferable that the reporter gene be selected from the group consisting of a gene encoding xcex2-glucuronidase, a gene encoding xcex2-galactosidase, a gene encoding luciferase, and a gene encoding fluorescence green protein. In an embodiment further still, a transgenic fungus is provided that comprises a reporter gene operatively linked to a constitutive promoter, which provides a control for the method. Preferably, the constitutive promoter is the promoter for the bena gene or mutant thereof.
Therefore, it is an object to provide a compound that inhibits transcription of one or more genes encoding proteins involved in secondary metabolism of fungi. In particular, compounds that inhibit genes involved in biosynthesis of mycotoxins.
It is also an object of the present invention to provide a method for determining whether an extract comprises compounds that inhibit transcription of one or more genes encoding a protein involved in secondary metabolism of fungi. In particular, compounds that inhibit genes involved in biosynthesis of mycotoxins.
Further still, it is an object of the present invention to provide a method for identifying and purifying compounds that inhibit transcription of one or more genes encoding a protein involved in secondary metabolism of fungi. In particular, compounds that inhibit genes involved in biosynthesis of mycotoxins.
These and other objects of the present invention will become increasingly apparent with reference to the following drawings and preferred embodiments.